Publications by authors named "Tsutomu Toki"

71 Publications

Dyserythropoietic anaemia with an intronic splicing mutation in patients suspected to have Diamond-Blackfan anaemia.

EJHaem 2022 Feb 10;3(1):163-167. Epub 2022 Jan 10.

Department of Pediatrics Hirosaki University Graduate School of Medicine Hirosaki Japan.

Diamond-Blackfan anaemia (DBA) shares clinical features with two recently reported sporadic cases of dyserythropoietic anaemia with a cryptic splicing mutation (c.871-24 C>T). We hypothesized that some patients clinically diagnosed with DBA but whose causative genes were unknown may carry the intronic mutation. Here, we examined 79 patients in our DBA cohort, who had no detectable causative genes. The intronic mutation was identified in two male patients sharing the same pedigree that included multiple cases with anaemia. Cosegregation of this mutation and disease in multiple family members provide evidence to support the pathogenicity of the intronic mutation.
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http://dx.doi.org/10.1002/jha2.374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9175706PMC
February 2022

Ribosomal protein L5 facilitates rDNA-bundled condensate and nucleolar assembly.

Life Sci Alliance 2022 07 23;5(7). Epub 2022 Mar 23.

Cancer Institute of Japanese Foundation for Cancer Research, Tokyo, Japan

The nucleolus is the site of ribosome assembly and formed through liquid-liquid phase separation. Multiple ribosomal DNA (rDNA) arrays are bundled in the nucleolus, but the underlying mechanism and significance are unknown. In the present study, we performed high-content screening followed by image profiling with the wndchrm machine learning algorithm. We revealed that cells lacking a specific 60S ribosomal protein set exhibited common nucleolar disintegration. The depletion of RPL5 (also known as uL18), the liquid-liquid phase separation facilitator, was most effective, and resulted in an enlarged and un-separated sub-nucleolar compartment. Single-molecule tracking analysis revealed less-constrained mobility of its components. rDNA arrays were also unbundled. These results were recapitulated by a coarse-grained molecular dynamics model. Transcription and processing of ribosomal RNA were repressed in these aberrant nucleoli. Consistently, the nucleoli were disordered in peripheral blood cells from a Diamond-Blackfan anemia patient harboring a heterozygous, large deletion in Our combinatorial analyses newly define the role of RPL5 in rDNA array bundling and the biophysical properties of the nucleolus, which may contribute to the etiology of ribosomopathy.
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http://dx.doi.org/10.26508/lsa.202101045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8942980PMC
July 2022

[New insights into inherited bone marrow failure syndrome].

Rinsho Ketsueki 2021 ;62(10):1455-1464

Department of Pediatrics, Hirosaki University Graduate School of Medicine.

Inherited bone marrow failure syndromes (IBMFS) are a heterogeneous group of genetic disorders characterized by bone marrow failure, congenital anomalies, and increased risk of malignant disease. Next generation sequencing methods have greatly facilitated the discovery of genetic etiology in IBMFS. Recently, de novo mutations activating TP53 were detected in patients with BMFS, mimicking Diamond-Blackfan anemia (DBA), using whole exome sequencing, and these patients were recognized as having a novel disorder. This discovery provides important insights into the previously postulated connection between p53 activation and IBMFS. Furthermore, a novel IBMFS, aldehyde degradation deficiency syndrome, was found in patients with aplastic anemia resembling Fanconi anemia (FA). This disorder is caused by combined inactivating mutations in ADH5 and ALDH2 coding formaldehyde-detoxifying enzymes. In this review, we highlight recent studies on DBA, FA, and their related diseases in Japan.
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http://dx.doi.org/10.11406/rinketsu.62.1455DOI Listing
November 2021

Enzymatic Changes in Red Blood Cells of Diamond-Blackfan Anemia.

Tohoku J Exp Med 2021 09;255(1):49-55

Department of Transfusion Medicine and Cell Processing, Faculty of Medicine, Tokyo Women's Medical University.

Diamond-Blackfan anemia is a congenital bone marrow failure syndrome characterized by red blood cell (RBC) aplasia with varied malformations in infants. Elevated activity of adenosine deaminase (ADA) has been considered as a useful biomarker of Diamond-Blackfan anemia, and ADA assay has been shown to be more sensitive than genetic diagnosis. Approximately, 80% of the examined patients showed elevated ADA activity, whereas genetic tests of ribosome subunit genes identified mutations in approximately 60% of the patients. We previously reported that reduced glutathione (GSH) levels in RBCs may serve as a biomarker of Diamond-Blackfan anemia. In this study, to confirm the universality of our data, we extended the analysis to seven RBC enzymes and GSH of 14 patients with Diamond-Blackfan anemia and performed a cross-analysis study using enzyme activity assay and recently reported proteome data. Statistical analysis revealed that both data exhibited high similarity, upregulation in the hexokinase and pentose-phosphate pathway, and downregulation in glycolytic enzymes such as phosphofructokinase and pyruvate kinase, in the RBCs obtained from the subjects with Diamond-Blackfan anemia. The only discrepancy between enzyme activity and proteome data was observed in glucose-6-phosphate dehydrogenase (G6PD), as increased G6PD activity showed no relation with the significant elevation in protein levels. These results suggest that our enzymatic activity data of Diamond-Blackfan anemia are universal and that the enzymatic activation of G6PD via a hitherto-unveiled mechanism is another metabolic feature of RBCs of Diamond-Blackfan anemia.
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http://dx.doi.org/10.1620/tjem.255.49DOI Listing
September 2021

A Case of Congenital Leukemia With MYB-GATA1 Fusion Gene in a Female Patient.

J Pediatr Hematol Oncol 2022 01;44(1):e250-e252

Department of Pediatrics, Hirosaki University Graduate School of Medicine, Aomori, Japan.

We report a female newborn with acute myelogenous leukemia (AML) associated with a MYB-GATA1 fusion gene. Morphologic findings of myeloid lineage were obtained using light microscopy. Cytogenetic analysis of peripheral blood showed a complex karyotype: 46,X,-X,add(3)(q21),der(6)add(6)(q21)del(6)(q?), +mar1[5]/46,XX[15]. Targeted RNA sequencing revealed a MYB-GATA1 fusion gene. Reduced-dose AML-type chemotherapy resulted in remission and survival for >3 years without relapse. The present case demonstrated the feasibility of carrying out targeted RNA sequencing for identifying MYB-GATA1 and supports the notion that neonatal AML with MYB-GATA1 with reduced chemotherapy may show better prognosis than other highly toxic therapies.
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http://dx.doi.org/10.1097/MPH.0000000000002119DOI Listing
January 2022

Association of Multiple Gene Polymorphisms Including Homozygous NUDT15 R139C With Thiopurine Intolerance During the Treatment of Acute Lymphoblastic Leukemia.

J Pediatr Hematol Oncol 2021 11;43(8):e1173-e1176

Community Medicine, Hirosaki University Graduate School of Medicine, Hirosaki.

Although thiopurine is a crucial drug for treating acute lymphoblastic leukemia, individual variations in intolerance are observed due to gene polymorphisms. A 3-year-old boy with B-cell precursor acute lymphoblastic leukemia who was administered thiopurine developed mucositis, sepsis, and hemophagocytic lymphohistiocytosis due to prolonged hematologic toxicity, chronic disseminated candidiasis, and infective endocarditis that triggered multiple brain infarctions. The patient was found to harbor 3 gene polymorphisms associated with thiopurine intolerance including homozygous NUDT15 R139C, heterozygous ITPA C94A, and homozygous MTHFR C677T and heterozygous RFC1 G80A. Thus, the combined effect of intolerance via multiple gene polymorphisms should be considered in case of unexpected adverse reactions.
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http://dx.doi.org/10.1097/MPH.0000000000002085DOI Listing
November 2021

Usefulness of functional splicing analysis to confirm precise disease pathogenesis in Diamond-Blackfan anemia caused by intronic variants in .

Pediatr Hematol Oncol 2021 Sep 24;38(6):515-527. Epub 2021 Feb 24.

Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan.

Diamond-Blackfan anemia (DBA) is mainly caused by pathogenic variants in ribosomal proteins and 22 responsible genes have been identified to date. The most common causative gene of DBA is [NM_001022.4]. Nearly 180 variants have been reported, including three deep intronic variants outside the splicing consensus sequence (c.72-92A > G, c.356 + 18G > C, and c.411 + 6G > C). We also identified one case with a c.412-3C > G intronic variant. Without conducting transcript analysis, the pathogenicity of these variants is unknown. However, it is difficult to assess transcripts because of their fragility. In such cases, in functional splicing assays can be used to assess pathogenicity. Here, we report functional splicing analysis results of four deep intronic variants identified in our case and in previously reported cases. One splicing consensus variant (c.411 + 1G > A) was also examined as a positive control. Aberrant splicing with a 2-bp insertion between exons 5 and 6 was identified in the patient samples and minigene assay results also identified exon 6 skipping in our case. The exon 6 skipping transcript was confirmed by further evaluation using quantitative RT-PCR. Additionally, minigene assay analysis of three reported deep intronic variants revealed that none of them showed aberrant splicing and that these variants were not considered to be pathogenic. In conclusion, the minigene assay is a useful method for functional splicing analysis of inherited disease.
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http://dx.doi.org/10.1080/08880018.2021.1887984DOI Listing
September 2021

Post-induction MRD by FCM and GATA1-PCR are significant prognostic factors for myeloid leukemia of Down syndrome.

Leukemia 2021 09 15;35(9):2508-2516. Epub 2021 Feb 15.

Division of Leukemia and Lymphoma, Children's Cancer Center, National Center for Child Health and Development, Tokyo, Japan.

Myeloid leukemia of Down syndrome (ML-DS) is associated with good response to chemotherapy, resulting in favorable outcomes. However, no universal prognostic factors have been identified to date. To clarify a subgroup with high risk of relapse, the role of minimal residual disease (MRD) was explored in the AML-D11 trial by the Japanese Pediatric Leukemia/Lymphoma Study Group. MRD was prospectively evaluated at after induction therapy and at the end of all chemotherapy, using flow cytometry (FCM-MRD) and GATA1-targeted deep sequencing (GATA1-MRD). A total of 78 patients were eligible and 76 patients were stratified to the standard risk (SR) group by morphology. In SR patients, FCM-MRD and GATA1-MRD after induction were positive in 5/65 and 7/59 patients, respectively. Three-year event-free survival (EFS) and overall survival (OS) rates were 95.0% and 96.7% in the FCM-MRD-negative population, and 60.0% and 80.0% in the positive population. Three-year EFS and OS rates were both 98.1% in the GATA1-MRD-negative population, and 57.1% and 71.4% in the positive population. Adjusted hazard ratios for associations of FCM-MRD with EFS were 14.67 (p = 0.01). Detection of MRD by either FCM or GATA1 after initial induction therapy represents a significant prognostic factor for predicting ML-DS relapse.
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http://dx.doi.org/10.1038/s41375-021-01157-wDOI Listing
September 2021

Reduced-intensity conditioning is effective for hematopoietic stem cell transplantation in young pediatric patients with Diamond-Blackfan anemia.

Bone Marrow Transplant 2021 05 18;56(5):1013-1020. Epub 2020 Sep 18.

Department of Pediatrics, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative therapy for the hematologic manifestations of Diamond-Blackfan anemia (DBA). However, data regarding the optimal conditioning regimen for DBA patients are limited. We retrospectively compared the outcomes of DBA patients who underwent HSCT using either myeloablative conditioning (MAC) or reduced-intensity conditioning (RIC) regimens. The patients belonged to a cohort treated at our hospitals between 2000 and 2018. HSCT was performed in 27 of 165 patients (16.4%). The median age at the time of HSCT was 3.6 years. Stem cell sources included bone marrow for 25 patients (HLA-matched sibling donors, n = 5; HLA-mismatched related donors, n = 2; HLA-matched/mismatched unrelated donors, n = 18) or cord blood for 2 patients. MAC or RIC regimens were used in 12 and 15 patients, respectively. Engraftment was successful in all 27 patients who underwent HSCT. Three patients who underwent HSCT using MAC regimens developed sinusoidal obstruction syndrome. The 3-year overall survival (OS) and failure-free survival rates (FFS) post-transplantations were 95.2% and 88.4%, respectively, with no significant differences between MAC and RIC regimens. Our data suggest that HSCTs using RIC regimens are effective and obtain engraftment with excellent OS and FFS for young DBA patients.
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http://dx.doi.org/10.1038/s41409-020-01056-1DOI Listing
May 2021

Outcome of colorectal cancer in Diamond-Blackfan syndrome with a ribosomal protein S19 mutation.

Clin J Gastroenterol 2020 Dec 8;13(6):1173-1177. Epub 2020 Jul 8.

Department of Clinical Oncology, Graduate School of Medicine, Akita University, Hirosaki University Graduate School of Medicine, Akita, Japan.

Diamond-Blackfan anemia is an autosomal dominant syndrome, characterized by anemia and a predisposition for malignancies. Ribosomal proteins are responsible for this syndrome, and the incidence of colorectal cancer in patients with this syndrome is higher than the general population. This patient's Diamond-Blackfan anemia was caused by a novel ribosomal protein S19 gene mutation, and he received chemotherapy for colorectal cancer caused by it. In his cancer, ribosomal proteins S19 and TP53 were overexpressed. He received 5FU and cetuximab; however, his anemia made chemotherapy difficult, and he did not survive long. Patients with Diamond-Blackfan anemia should be screened earlier and more often for colorectal cancer than usual.
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http://dx.doi.org/10.1007/s12328-020-01176-7DOI Listing
December 2020

Clinical, cytogenetic, and molecular analyses of 17 neonates with transient abnormal myelopoiesis and nonconstitutional trisomy 21.

Pediatr Blood Cancer 2020 04 5;67(4):e28188. Epub 2020 Feb 5.

Department of Pediatrics, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.

Background: Transient abnormal myelopoiesis (TAM) is a unique myeloproliferative disorder that occurs in neonates with constitutional trisomy 21/Down syndrome (DS). Although TAM also develops in neonates without constitutional trisomy 21, the clinical, cytogenetic, and molecular characteristics of those patients are not fully understood.

Procedure: We retrospectively evaluated the clinical and cytogenetic findings and GATA1 mutation status of 17 neonates with TAM and nonconstitutional trisomy 21 tested for GATA1 mutations at our institute, and compared the findings with those of 64 neonates with TAM and constitutional trisomy 21/DS.

Results: DS clinical features were observed in five of the 17 (29%) patients. In all patients, both trisomy 21 and GATA1 mutations were detected in diagnostic samples. Over a median follow-up of 33 (range, 0-139) months, early death (< 6 months of age) occurred in four patients (24%). Overall and event-free survivals were not significantly different between the patients with TAM and nonconstitutional trisomy 21 and those with TAM and constitutional trisomy 21/DS (five-year overall survival: 76% ± 10% vs 53% ± 13%, P = 0.40; five-year event-free survival: 55% ± 13% vs 48% ± 12%, P = 0.90). The five-year cumulative incidence of progression to myeloid leukemia of DS was also similar between the groups (21% vs 24%, P = 0.80).

Conclusions: Patients with TAM and nonconstitutional trisomy 21 exhibited similar biology and outcomes to those with TAM and constitutional trisomy 21/DS. The possibility of TAM should be considered even in phenotypically normal neonates with TAM symptoms, for appropriate management.
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http://dx.doi.org/10.1002/pbc.28188DOI Listing
April 2020

Highly sensitive detection of GATA1 mutations in patients with myeloid leukemia associated with Down syndrome by combining Sanger and targeted next generation sequencing.

Genes Chromosomes Cancer 2020 03 21;59(3):160-167. Epub 2019 Oct 21.

Department of Pediatrics, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.

Myeloid leukemia associated with Down syndrome (ML-DS) is characterized by a predominance of acute megakaryoblastic leukemia, the presence of GATA1 mutations and a favorable outcome. Because DS children can also develop conventional acute myeloid leukemia with unfavorable outcome, detection of GATA1 mutations is important for diagnosis of ML-DS. However, myelofibrosis and the significant frequency of dry taps have hampered practical screening of GATA1 mutations using bone marrow (BM) samples. In response to those problems, 82 patients were enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group AML-D11 study. GATA1 mutations were analyzed by Sanger sequencing (SS) using genomic DNA (gDNA) from BM and cDNA from peripheral blood (PB) followed by targeted next-generation sequencing (NGS) using pooled diagnostic samples. BM and PB samples were obtained from 71 (87%) and 82 (100%) patients, respectively. GATA1 mutations were detected in 46 (56%) and 58 (71%) patients by SS using BM gDNA and PB cDNA, respectively. Collectively, GATA1 mutations were identified in 73/82 (89%) patients by SS. Targeted NGS detected GATA1 mutations in 74/82 (90%) patients. Finally, combining the results of SS with those of targeted NGS, GATA1 mutations were identified in 80/82 (98%) patients. These results indicate that SS using BM gDNA and PB cDNA is a rapid and useful method for screening for GATA1 mutations in ML-DS patients. Thus, a combination of SS and targeted NGS is a sensitive and useful method to evaluate the actual incidence and clinical significance of GATA1 mutations in ML-DS patients.
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http://dx.doi.org/10.1002/gcc.22816DOI Listing
March 2020

Integrated genetic and epigenetic analysis revealed heterogeneity of acute lymphoblastic leukemia in Down syndrome.

Cancer Sci 2019 Oct 10;110(10):3358-3367. Epub 2019 Sep 10.

Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Children with Down syndrome (DS) are at a 20-fold increased risk for acute lymphoblastic leukemia (ALL). Compared to children with ALL and no DS (non-DS-ALL), those with DS and ALL (DS-ALL) harbor uncommon genetic alterations, suggesting DS-ALL could have distinct biological features. Recent studies have implicated several genes on chromosome 21 in DS-ALL, but the precise mechanisms predisposing children with DS to ALL remain unknown. Our integrated genetic/epigenetic analysis revealed that DS-ALL was highly heterogeneous with many subtypes. Although each subtype had genetic/epigenetic profiles similar to those found in non-DS-ALL, the subtype distribution differed significantly between groups. The Philadelphia chromosome-like subtype, a high-risk B-cell lineage variant relatively rare among the entire pediatric ALL population, was the most common form in DS-ALL. Hypermethylation of RUNX1 on chromosome 21 was also found in DS-ALL, but not non-DS-ALL. RUNX1 is essential for differentiation of blood cells, especially B cells; thus, hypermethylation of the RUNX1 promoter in B-cell precursors might be associated with increased incidence of B-cell precursor ALL in DS patients.
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http://dx.doi.org/10.1111/cas.14160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6778645PMC
October 2019

Resistance of t(17;19)-acute lymphoblastic leukemia cell lines to multiagents in induction therapy.

Cancer Med 2019 Sep 15;8(11):5274-5288. Epub 2019 Jul 15.

Department of Pediatrics, School of Medicine, University of Yamanashi, Chuo, Japan.

t(17;19)(q21-q22;p13), responsible for TCF3-HLF fusion, is a rare translocation in childhood B-cell precursor acute lymphoblastic leukemia(BCP-ALL). t(1;19)(q23;p13), producing TCF3-PBX1 fusion, is a common translocation in childhood BCP-ALL. Prognosis of t(17;19)-ALL is extremely poor, while that of t(1;19)-ALL has recently improved dramatically in intensified chemotherapy. In this study, TCF3-HLF mRNA was detectable at a high level during induction therapy in a newly diagnosed t(17;19)-ALL case, while TCF3-PBX1 mRNA was undetectable at the end of induction therapy in most newly diagnosed t(1;19)-ALL cases. Using 4 t(17;19)-ALL and 16 t(1;19)-ALL cell lines, drug response profiling was analyzed. t(17;19)-ALL cell lines were found to be significantly more resistant to vincristine (VCR), daunorubicin (DNR), and prednisolone (Pred) than t(1;19)-ALL cell lines. Sensitivities to three (Pred, VCR, and l-asparaginase [l-Asp]), four (Pred, VCR, l-Asp, and DNR) and five (Pred, VCR, l-Asp, DNR, and cyclophosphamide) agents, widely used in induction therapy, were significantly poorer for t(17;19)-ALL cell lines than for t(1;19)-ALL cell lines. Consistent with poor responses to VCR and DNR, gene and protein expression levels of P-glycoprotein (P-gp) were higher in t(17;19)-ALL cell lines than in t(1;19)-ALL cell lines. Inhibitors for P-gp sensitized P-gp-positive t(17;19)-ALL cell lines to VCR and DNR. Knockout of P-gp by CRISPRCas9 overcame resistance to VCR and DNR in the P-gp-positive t(17;19)-ALL cell line. A combination of cyclosporine A with DNR prolonged survival of NSG mice inoculated with P-gp-positive t(17;19)-ALL cell line. These findings indicate involvement of P-gp in resistance to VCR and DNR in Pgp positive t(17;19)-ALL cell lines. In all four t(17;19)-ALL cell lines, RAS pathway mutation was detected. Furthermore, among 16 t(1;19)-ALL cell lines, multiagent resistance was usually observed in the cell lines with RAS pathway mutation in comparison to those without it, suggesting at least a partial involvement of RAS pathway mutation in multiagent resistance of t(17;19)-ALL.
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http://dx.doi.org/10.1002/cam4.2356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6718581PMC
September 2019

MYH7 mutation identified by next-generation sequencing in three infant siblings with bi-ventricular noncompaction presenting with restrictive hemodynamics: A report of three siblings with a severe phenotype and poor prognosis.

J Cardiol Cases 2019 Apr 11;19(4):140-143. Epub 2019 Feb 11.

Department of Pediatrics, Hirosaki University School of Medicine and Graduate School of Medicine, Hirosaki, Japan.

Noncompaction of the ventricular myocardium (NVM) is a genetically heterogeneous cardiomyopathy. Various mutations associated with NVM have been identified in several genes. NVM patients usually present with complications of dilated cardiomyopathy. We identified a missense mutation, c.5740G>A, p.Glu1914Lys of , by targeted next-generation sequencing in three infant siblings with isolated bi-ventricular noncompaction who presented with restrictive hemodynamics and severe clinical courses. This mutation appears to be associated with a severe phenotype and poor prognosis. Early heart transplantation should be considered in similar cases. < No clear noncompaction of the ventricular myocardium genotype-phenotype correlations have been found to predict the clinical course. This report describes a mutation in three infant siblings with isolated bi-ventricular noncompaction who presented with restrictive hemodynamics and severe clinical courses. Early heart transplantation should be considered in cases with a similar genotype and/or phenotype.>.
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http://dx.doi.org/10.1016/j.jccase.2018.12.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451088PMC
April 2019

Two siblings with familial neuroblastoma with distinct clinical phenotypes harboring an ALK germline mutation.

Genes Chromosomes Cancer 2018 12 22;57(12):665-669. Epub 2018 Oct 22.

Department of Pediatrics, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.

The authors report two siblings with familial neuroblastoma with a germline R1275Q mutation of the tyrosine kinase domain of ALK. Whole exome sequencing and copy number variation assay were performed to investigate genetic alterations in the two cases. No common somatic mutations or gene polymorphisms related to the tumorigenesis of neuroblastoma were detected. A distinct pattern involving both segmental chromosomal alteration and MYCN amplification was detected. The diversity of biological behavior of familial neuroblastoma harboring a germline ALK mutation may depend on conventional prognostic factors, such as segmental chromosomal alterations and MYCN amplification, rather than additional acquired mutations.
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http://dx.doi.org/10.1002/gcc.22676DOI Listing
December 2018

De Novo Mutations Activating Germline TP53 in an Inherited Bone-Marrow-Failure Syndrome.

Am J Hum Genet 2018 09 23;103(3):440-447. Epub 2018 Aug 23.

Department of Transfusion Medicine and Cell Processing, Tokyo Women's Medical University, Tokyo 162-8666, Japan.

Inherited bone-marrow-failure syndromes (IBMFSs) include heterogeneous genetic disorders characterized by bone-marrow failure, congenital anomalies, and an increased risk of malignancy. Many lines of evidence have suggested that p53 activation might be central to the pathogenesis of IBMFSs, including Diamond-Blackfan anemia (DBA) and dyskeratosis congenita (DC). However, the exact role of p53 activation in each clinical feature remains unknown. Here, we report unique de novo TP53 germline variants found in two individuals with an IBMFS accompanied by hypogammaglobulinemia, growth retardation, and microcephaly mimicking DBA and DC. TP53 is a tumor-suppressor gene most frequently mutated in human cancers, and occasional germline variants occur in Li-Fraumeni cancer-predisposition syndrome. Most of these mutations affect the core DNA-binding domain, leading to compromised transcriptional activities. In contrast, the variants found in the two individuals studied here caused the same truncation of the protein, resulting in the loss of 32 residues from the C-terminal domain (CTD). Unexpectedly, the p53 mutant had augmented transcriptional activities, an observation not previously described in humans. When we expressed this mutant in zebrafish and human-induced pluripotent stem cells, we observed impaired erythrocyte production. These findings together with close similarities to published knock-in mouse models of TP53 lacking the CTD demonstrate that the CTD-truncation mutations of TP53 cause IBMFS, providing important insights into the previously postulated connection between p53 and IBMFSs.
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http://dx.doi.org/10.1016/j.ajhg.2018.07.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6128301PMC
September 2018

[Diagnostic targets and exosome sequence analysis of Diamond-Blackfan anemia in Japan].

Rinsho Ketsueki 2018;59(7):945-952

Department of Pediatrics, Hirosaki University Graduate School of Medicine.

Diamond-Blackfan anemia (DBA) is a rare, inherited, congenital bone marrow failure syndrome, which typically manifests in infancy. In addition, the disease is often accompanied by normochromic macrocytic anemia and acute reticulocytopenia in the peripheral blood and by selective deficiency of erythroid precursors in the normocellular bone marrow. Approximately 50% of patients with DBA exhibit growth retardation and multiple congenital anomalies, which primarily include craniofacial dysmorphism, upper-limb abnormalities, urogenital malformations, and congenital heart defects. In addition, heterozygous mutations in genes encoding ribosomal proteins (RP) that lead to a loss of function of the genes are detected in nearly 60% of patients with DBA. As a majority of identified causative genes belong to RP genes, the impairment of ribosome biogenesis is considered accountable for the disease. This study aims to outline the molecular pathology of DBA and the causative gene RPS15A isolated using our exosome analysis.
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http://dx.doi.org/10.11406/rinketsu.59.945DOI Listing
July 2019

Correction to: Atypical erythroblastosis in a patient with Diamond-Blackfan anemia who developed del(20q) myelodysplasia.

Int J Hematol 2018 08;108(2):236

Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

The corresponding author should be ''Masataka Ishimura'', and not ''Motoshi Sonoda'' as given in the original publication of the article.
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http://dx.doi.org/10.1007/s12185-018-2493-4DOI Listing
August 2018

Atypical erythroblastosis in a patient with Diamond-Blackfan anemia who developed del(20q) myelodysplasia.

Int J Hematol 2018 Aug 23;108(2):228-231. Epub 2018 Feb 23.

Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia arising from ribosomal protein (RP) defects. Affected patients present with neonatal anemia, occasional dysmorphism, and cancer predisposition. An anemic newborn was diagnosed with DBA due to RPL5 mutation (c.473_474del, p.K158SfsX26). Refractory anemia required regular transfusions and iron chelation therapy. Pancytopenia occurred at age 16 years. Bone-marrow studies showed myelodysplasia, erythroblastosis, and clonal evolution of del(20)(q11.2q13.3). Severe anemia required transfusions. Del(20q), including the L3MBTL1 gene, is reported to be relevant to the hematological phenotype of Shwachman-Diamond syndrome. A combined defect of RPL5 and L3MBTL1 may contribute to the aberrant erythropoiesis in the present case.
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http://dx.doi.org/10.1007/s12185-018-2424-4DOI Listing
August 2018

Purification of leukemic blast cells from blood smears using laser microdissection.

Int J Hematol 2017 Jul 13;106(1):55-59. Epub 2017 Apr 13.

Department of Human Health Sciences, Graduate School of Medicine, Kyoto University, 53 Kawahara-cho, Syogoin, Sakyoku, Kyoto, 606-8507, Japan.

In treatment of acute myeloid leukemia (AML), prognostic factors, including gene mutation and abnormal gene expression, enable risk stratification of patients. However, in the case of a small proportion of leukemic blast cells, such as AML associated with Down syndrome (AML-DS), it is not possible to examine prognostic factors precisely due to the large proportion of normal cells. Here, we present a novel method for examining prognostic factors by making a smear on a membrane slide glass from a small amount of diagnostic specimen and collecting highly pure leukemic blast cells by laser microdissection (LMD). We verified the effectiveness of this method using 10% KPAM1 cell line suspension and peripheral blood containing 20% blast cells obtained from a patient with transient abnormal myelopoiesis (TAM). After making blood smears, approximately 100 cells were collected and analyzed by direct sequencing. Frameshift mutations (2 bp deletion and 17 bp duplication, respectively) in GATA-1 were detected in each sample, suggesting KPAM1 and TAM blast cells were accurately purified. This novel method enables us to precisely examine prognostic factors in many cases, even in cases with a small proportion of leukemic blast cells or small specimens to preserve.
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http://dx.doi.org/10.1007/s12185-017-2227-zDOI Listing
July 2017

Diagnostic challenge of Diamond-Blackfan anemia in mothers and children by whole-exome sequencing.

Int J Hematol 2017 Apr 23;105(4):515-520. Epub 2016 Nov 23.

Department of Pediatrics, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-kogushi, Ube, Yamaguchi, 755-8505, Japan.

Diamond-Blackfan anemia (DBA) is a pure red cell aplasia that arises from defective ribosomal proteins (RPs). Patients with this rare ribosomopathy present with neonatal anemia and occasional dysmorphism. Clinical heterogeneity and clusters of causative RP genes hamper the diagnosis and perinatal management. We report three mother-and-child pairs of anemia who were finally diagnosed by whole-exome sequencing. Each pair showed distinct disease severity and response to anemia treatment. Only one mother had the diagnostic dysmorphism, including short stature, webbed neck, and thenar hypoplasia. This mother had a frame-shift mutation of RPL11 (exon 3, c.58_59del). Her infant showed transient neonatal anemia, but had no mutations of RP genes. The other mother-child pairs had a missense mutation of RPS19 (exon 4, c.185G>A), and a splicing error of RPS7 (exon 3, c.76-1G>T), respectively. Other than the reported mutations, there were no variants in genes significantly associated with anemia. Our results suggested that whole-exome sequencing (WES) is effective for achieving a prompt and correct diagnosis of human ribosomopathy.
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http://dx.doi.org/10.1007/s12185-016-2151-7DOI Listing
April 2017
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